Stat (Signal Transducers and Activators of Transcription) proteins are latent transcription factors that become activated by phosphorylation on a single tyrosine (near residue 700), typically in response to extracellular ligands (Darnell, 1997; Stark et al., 1998; U.S. application Ser. Nos. 08/212,185, filed Mar. 11, 1994; Ser. No. 09/087,465, filed May 29, 1998; Ser. No. 08/951,130, filed Oct. 15, 1997; Ser. No. 09/012,710, filed Jan. 23, 1998; Ser. No. 08/820,754, filed Mar. 19, 1997; and U.S. Pat. No. 5,716,622; all of the foregoing incorporated herein by reference in their entireties). More than 40 different polypeptide ligands cause Stat phosphorylation through either cytokine receptors plus associated Jak kinases or growth factors (e.g. EGF, PDGF, CSF-1) acting through intrinsic receptor tyrosine kinases. An active Stat dimer is formed via the reciprocal interactions between the SH2 domain of the monomer and the phosphorylated tyrosine of the other (Chen et al., 1998). The dimers accumulate in the nucleus, recognize specific DNA elements and activate transcription. The Stat proteins are subsequently inactivated by tyrosine dephosphorylation and return to the cytoplasm (Haspel et al., 1996).
Ligand dependent activation of the Stats is often associated with differentiation and/or growth regulation while constitutive activation of Stats (i.e. activation without known requirements of extracellular polypeptides) is often associated with growth dysregulation. For example, the development of the antiviral state or growth restraint secondary to either IFN-.alpha. of IFN-.gamma. treatment requires transcriptionally competent Stat1 (Bromberg et al., 1996; Horvath and Darnell, 1996) and mice lacking Stat1 form chemically induced tumors of the skin more easily than wild-type animals (Kaplan et el., 1998). Various stages of lymphocyte or monocyte development depend on Stats 3, 4 and 6 (Kaplan et al., 1996; Takeda et al., 1996; Thierfelder et al., 1996; Takeda et al., 1998; Takeda et al., 1999); development of breast epithelium requires Stat5A (Liu et al., 1997; Teglund et al., 1998), and proper male growth hormone response demands Stat5B (Udy et al, 1997; Teglund et al., 1998). A growing number of tumor-derived cell lines as well as samples from human cancers are reported to contain constitutively activated Stat proteins, very frequently Stat3 (Garcia and Jove, 1998). For example, all src transformed cell lines exhibit constitutively activated Stat3 (Yu et al, 1995). Moreover, dominant negative Stat3 suppresses src transformation without having any effect on ras transformation (Bromberg et al., 1998; Turkson et al., 1998). Cell lines from multiple myelomas that have become growth factor independent require constitutively active Stat3 to protect against apoptosis (Catlett-Falcone et al., 1999). A high proportion of head and neck cancers (often of squamous cell origin) have constitutively active Stat3, most likely secondary to aberrant EGF receptor signaling (Grandis et al., 1998) and dominant negative Stat3 slows the growth of cell lines developed from these cancers.
Oncogenes were first defined as viral or mutated cellular genes that could confer a transformed phenotype to cultured cells (Lodish et al., 1995). When oncogenes were characterized at the molecular level, many were found to be constitutively activated. One constitutively activated Stat molecule has been described. Amino acid changes in two separate domains of Stat5 results in constitutive activation and obviates the need for IL-3 in the growth of BaF3 cells (Onishi et al., 1998). However, the amino acids that are changed in the Stat5 molecule (H-R.sup.299 and S-F.sup.711) are not conserved between Stat5 and Stat3.
It is toward the development of a dimerizable and constitutively active Stat protein particularly in the absence of tyrosine phosphorylation and its utility in investigating the role of the active protein in transcription and inducing cell transformation, and the effects of modulators thereon, that the present invention is directed.
The citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the instant application.